Rolling tubes and T-flasks demonstrated comparable culture performance and metabolite profiles, as well as product potencies
(Figures 1, 2, and 3). In comparison, 6-well plates demonstrated inferior growth to either T75 flasks or 50-mL rolling tubes.
Cell density (Figure 1) reached a maximum of ~0.7 x 106 vc/mL compared with ~2.0 x 106 vc/mL for the other two platforms. Glutamine consumption (Figure 2a) was lower, although no significant difference in glucose
consumption was observed (Figure 2c). Lactate levels increased (Figure 2d) possibly because of less efficient gas transfer
in the 6-well platform, causing a shift toward anaerobic metabolism. The significant increase in osmolality over time (Figure
2h) was notable. This phenomenon was investigated using 6-well plates with uninoculated media.
Evaporation was the apparent reason for the significant rise in osmolality and in glutamine and glucose metabolites in the
6-well plate platform (data not shown). Total production of the desired protein (Figure 3a) also was much lower in 6-well
plates than in the other platforms, though per cell specific productivity (Figure 3b) was ~40% greater. This could have resulted
from the much lower viable cell densities (Figure 1a) in the 6-well plate platform caused by the higher osmolality resulting
from evaporation or a shift in metabolism toward production (rather than growth) because of osmotic stress.4
The performance of the small-scale systems also was representative of commercial production on the basis of spent media analysis
performed on cultures from both scales using the same cell line and media. It should be noted that seeding density and passage
schedule have been designed to emulate cell-specific perfusion rate in production bioreactors. In Figure 4, the amino acid
profile of small scale is compared to production-scale culture. The similarity in the profile (i.e., in the extent of consumption,
or for some amino acids, the extent of production) between the scales strongly suggests that the optimized small-scale systems
are able to predict the performance in bioreactors in applications such as media or clone screening.
Figure 4. Amino acid analysis of small scale versus production-scale spent media. The percent change in levels of different
amino acids in spent media relative to the starting fresh media is shown for production scale runs and for representative
screening scale runs (T-flask).
In this study, we assessed the performance of the rolling tube platform compared to the T-75 flask and the 6-well plate platforms.
Although T-flasks recently have replaced the large roller bottles in many laboratory-scale applications on the basis of culture
performance (internal data, not shown), they also have major drawbacks including throughput and ease of operation. Other small-scale
systems used in a variety of screening applications, such as micro-bioreactors, are not covered here because they have major
drawbacks, most notably cost.5 This study demonstrates that the rolling-tube system is a platform of choice for development projects because it performs
similarly to the T-flask (and the roller bottle, data not shown) but is much more user friendly and has higher throughput.
Evaporation minimization, pH control, and gas transfer rates are superior in the rolling tube system as a result of the mixing
through rolling motion where media is in greater contact with the vessel surface and because of the geometry of the tubes
that contain vented caps.
On the other hand, the 6-well plate platform demonstrated inferior performance resulting from evaporation and gas transfer
problems. Data including metabolite analysis, cell growth rate, viability, specific productivity, and product titer and potency
show the performance of rolling tubes is comparable to the T-flask and superior to 6-well plates. The rolling tubes also demonstrated
robust and consistent performance at a broad range of working volumes. This allows for the designing of fill volumes tailored
to the volumes and frequency required for sampling (e.g., for metabolites and titer determination) in a given experimental
and screening design. Seeding density and passage schedule have been optimized to emulate perfusion reactor conditions.
Samples submitted to spent media analysis (e.g., amino acids, Figure 4) demonstrated a similar profile to that of production
samples, indicating scalability and the ability to predict performance in production bioreactors. The platform is ideal for
suspension-adapted cell lines because the cell tested did not adhere to the tube's surface, and because passaging and harvest
is greatly facilitated by the possibility of using the growth vessels (i.e., the 50-mL tube) as the harvest vessel by centrifugation,
eliminating volume transfer steps. This significantly reduces labor handling and the risk of contamination, and increases
efficiency. Ergonomics and handling also are made easier by the ability to line up tubes in a relatively small footprint inside
a biological hood; volume culture transfer is facilitated by the wide opening of the tubes.
Additional benefits of the rolling-tube system include: low material and operation costs (for purchase of the disposable tubes
and rolling device), low media volume usage, and the ability to run up to 16 tubes per platform simultaneously; this number
can be increased by using additional stackable platforms. The platforms can be placed on a shelf inside a humidified temperature
controlled CO2 incubator. The platform withstands continuous operation in the incubator's environment. Given the advantages of the rolling-tube
system in performance and handling, it is a preferred platform for a variety of cell culture applications.
The rolling-tube platform presents an ideal balance between predictability of performance at production scale, throughput,
robustness, and ease of use, while also providing sufficient sample volumes for product and analytical testing. Its gentle
agitation with mixing through rolling also emulates the cumbersome but traditional roller bottle "small-scale" system. We
currently use the rolling-tube platform in cell line selection and process development projects such as media screening using
design of experiment approaches.
Yuval Shimoni is principal engineer, Carmen Chin is a senior associate process engineer II, Teng Liu is a laboratory technician, Veronica Hernandez-Rodriguez is a laboratory assistant, Peter Kramer is director, and Jin Wang is manager, all in manufacturing sciences, product supply biotech at Bayer HealthCare, Berkeley, CA, 510.705.5775, firstname.lastname@example.org
Volker Moehrle is a senior scientist at Bayer Technology Services, Leverkusen, Germany.
1. Eibl R, Kaiser S, Lombriser R, Eibl D. Disposable bioreactors: the current state-of-the-art and recommended applications
in biotechnology. Appl Microbiol Biotechnol. 2010;86:41–9.
2. De Jesus MJ, Girard P, Bourgeois M, Baumgartner G, Jacko B, Amstutz H, Wurm FM. TubeSpin satellites: A fast track approach
for process development with animal cells using shaking technology. Biochem Engin J. 2004;17:217–23.
3. Jordan M, Jenkins N. Tools for high-throughput medium and process optimization. Methods in Biotechnology: methods and
protocols, 2nd Ed. Portner R (Editor) 2007;24:193–202.
4. Yi X, Sun X, Zhang Y. Effects of osmotic pressure on recombinant BHK cell growth and von willebrand factor (vWF) expression.
Proc Biochem. 2004;39:1817–23.
5. Amanullah A, Otero JM, Mikola M, Hsu A, Zhang J, Aunins J, Schreyer HB, Hope J, Russo P. Novel micro-bioreactor high throughput
technology for cell culture process development: reproducibility and scalability assessment of fed-batch CHO cultures. Biotechnol